Effect Of Liraglutide On Diabetes-induced Cognitive Decline In Mice And Its Molecular Mechanisms

Diabetes mellitus is characterized by chronic hyperglycemia often accompanied by glycosuria,polydipsia,and polyuria,which is a significant global public health problem depicting a rising prevalence worldwide.As a serious but often overlooked complication of diabetes,diabetes-associated cognitive decline is attracting increasing attention.However,the underlying mechanisms and related clinical interventions and treatments available to prevent this disorder are yet to be fully determined.Autophagy has been reported to modulate neuronal survival and death and be associated with several neurodegenerative diseases.However,it is not clear whether autophagy is involved in diabetes-related cognitive decline.Given the widespread and serious influence of diabetes-induced cognitive impairments on individuals,exploring its underlying pathogenesis will help us to better understand this disease and develop more effective treatments or novel therapeutic targets for this disorder.The glucagon-like peptide-1 analog liraglutide has been well established to exert neuroprotective effects in various experimental animal models and neurodegenerative diseases.Nevertheless,the effect of liraglutide on diabetes-induced cognitive decline is still not very clear.In the current study,we investigated the potential molecular mechanisms of diabetes-associated cognitive decline and further explore possible interventions using a streptozotocin(STZ)-induced diabetic mouse model and primary cultured mouse hippocampal neurons.Our results indicate that diabetes induces neuronal injuries and following learning and memory impairments in an experimental mouse model of diabetes.And autophagy is involved in the process,which may play a protective role in diabetes-induced neuronal damages.Furthermore,we find that liraglutide ameliorates diabetes-induced hippocampal neuronal injuries and cognitive decline.Meanwhile,liraglutide further upreglulates autophagy level through AMPK/mTOR pathway.Therefore,We speculate that liraglutide exerts neuroprotective action against diabetes-induced hippocampal neuronal injuries and cognitive impairments by promoting autophagy via the AMPK/mTOR pathway.Taken together,the current study reveals the potential neuroprotective effect of liraglutide on diabetes-related cognitive decline and its related mechanisms.Our research was divided into two parts:Part One:Effect of Autophagy on Diabetes-induced Neuronal Injuries and Cognitive Decline.Part Two:Liraglutide ameliorates diabetes-induced cognitive decline by promoting autophagy via the AMPK/mTOR pathway.Part One Effect of Autophagy on Diabetes-induced Neuronal Injuries and Cognitive DeclineObjectives:Diabetes mellitus is a significant global public health problem depicting a rising prevalence worldwide.As a serious but often overlooked complication of diabetes,diabetes-associated cognitive decline is attracting increasing attention.However,the underlying mechanisms are yet to be fully detennined.Autophagy has been reported to modulate neuronal survival and death and be associated with several neurodegenerative diseases.In the current study,we aimed to investigate the possible role of autophagy in diabetes-induced neuronal injuries and cognitive impairments in vivo and vitro.Methods:Six-week-old male clean healthy C57BL/6 mice were divided into two groups randomly:the control group(Con,n＝15)and the STZ-injected group(DM,n＝15).Diabetic animal model was induced by intrapertoneal injection of one single dose of streptozotocin(STZ).Study of diabetes-induced cognitive decline:blood glucose,body weight,food intake and fluid intake were recorded after successful induction of diabetic animal model;eight weeks later,the cognitive function was examined by open-field test and Morris Water Maze test;morphological analysis was assessed by HE staining;changes in the number of neurons were evaluated by Nissl staining;ultrastructural changes of synapse and mitochondria were observed by transmission electron microscopy(TEM);Synaptophysin、PSD95、Cleaved caspase-3、Bcl-2 and Bax were determined by Western blot.Study of the role of autophagy in diabetes-induced neuronal injuries:expression levels of autophagy-associated proteins LC3、Beclin 1and p62 were examined by Western blot and double immunofluorescence labeling;the number of autophagic vacuoles was evaluated by TEM;hippocampal primary neurons and autophagy inhibitor Bafilomycin A1 were employed to further explore the role of autophagy in neuronal apoptosis.Results:During the experiment,DM mice showed significant changes in metabolic parameters,including significantly increased blood glucose level,decreased body weight and enhanced fluid and food intake(P<0.05).DM mice displayed deficits in spatial learning and memory capabilities as indicated by the longer escape latency to find the platform,the fewer times of original platform crossing and the less time spent in the target quadrant(P<0.05).In addition,DM mice exhibited obviously pathological changes in the neurons of hippocampal CA1 region,as indicated by irregular arrangement,widened intercellular space,reduced cell volume with condensation of nucleus and blurring of nucleolus.Moreover,a significantly decreased neuronal density in the hippocampal CA1 region was also detected in DM mice compared to Con mice(P<0.05).Meanwhile,TEM revealed that chemical synapses of the mice in the DM group were damaged,as manifested by decreased number of synapses,reduced synaptic vesicles,decreased postsynaptic region,deformed anterior region,a decreased postsynaptic density(PSD)area and a widened synaptic cleft.And mitochondrial structure was destroyed with disrupted or disappeared mitochondrial cristae.The protein expressions of Synaptophysin and PSD95 were decreased significantly in the mice of DM group compared with that of Con group(P<0.05).Furthermore,the results indicated an elevated level of autophagy in DM mice,as demonstrated by upregulated expression of autophagy markers LC3-II,Beclin 1 and downregulated level of p62,elevated percentage of LC3/NeuN/DAPI double-labeled neurons and increased formation of autophagic vacuoles(P<0.05),In vitro study,the high glucose-induced enhancement in the levels of apoptosis-related proteins was even more pronounced following Bafilomycin A1 pretreatment(P<0.05).Conclusions:The current study indicates that diabetes induces neuronal injuries and following damage of synaptic structure and function to impair the learning and memory in an experimental mouse model of diabetes.More important,autophagy was promoted by diabetes,which may play a protective role in diabetes-induced neuronal damages.Part Two Liraglutide ameliorates diabetes-induced cognitive decline by promoting autophagy via the AMPK/mTOR pathwayObjectives:Diabetic cognitive dysfunction has aroused a widely attention of the public for its widespread and profound influence on individuals with diabetes.However,there are few clinical interventions and treatments available to prevent this disorder.Liraglutide,a glucagon like peptide 1(GLP-1)analogs,promotes insulin secretion in response to the level of blood glucose.Liraglutide exerts beneficial effects at extra-pancreatic sites and has been well established to exert neuroprotective effects in various experimental animal models and neurodegenerative diseases.In the present study,we explored the effect of liraglutide on diabetes-induced neuronal apoptosis and cognitive impairments,and further explore the potential molecular mechanisms.Methods:Six-week-old male clean healthy C57BL/6 mice were divided into three groups randomly:the control group(Con,n=15),the STZ-injected group(DM,n=15)and the STZ+liraglutide-injected group.Liraglutide was injected intraperitoneally for eight weeks after successful induction of diabetic animal model.Study of effect of liraglutide on diabetes-induced cognitive decline:blood glucose,body weight,food intake and fluid intake were recorded during the experiment;eight weeks later,the cognitive function was examined by open-field test and Morris Water Maze test;changes in the morphology and number of neurons were observed by HE staining and Nissl staining;changes in synaptic ultrastructure were observed by TEM;expression ofCleaved caspase-3 was determined by Western blot.Study of the mechanisms of neuroprotective effect of liraglutide:expression levels of autophagy-associated genes Atg 12、Atg5、LC3、Beclin 1 and p62 were examined by Western blot and Real-time RT-PCR;aggregation of LC3 protein was determined by double immunofluorescence labeling;hippocampal primary neurons and a selective AMPK inhibitor Compound C were used in the current study to further explore the potential related mechanisms.Results:After STZ injection,DM mice presented typical metabolic characteristics of diabetes with high levels of blood glucose,reduced body weight and obviously polydipsia,polyphagia,polyuria.While after eight weeks of liraglutide treatment,DM+LRG mice did not show significant improvement in the above metabolic indices(P>0.05).Liraglutide treatment improved the increase in the escape latency to find the platform,the decrease in the times of original platform crossing and time spent in the target quadrant in DM mice(P<0.05).Additionally,liraglutide treatment significantly relieved the damage in neuronal morphological structure,the decrease in neuronal density,and the impairments in synaptic ultrastructure of DM mice in the hippocampal CA1 region.And the protein expression of Cleaved caspase-3 was decreased significantly in the mice of DM+LRG group compared with that of DM group(P<0.05).Furthermore,the results indicated an elevated level of autophagy both in DM mice and DM+LRG mice compared with Con mice,as demonstrated by upregulated expression levels of Atgl2,Atg5,LC3,Beclinl and downregulated level of p62,elevated percentage of LC3/NeuN/DAPI double-labeled neurons(P<0.05),while the trend was even more pronounced in DM+LRG mice(P<0.05).In vitro study,the results indicated that Compound C exhibited an inhibitory effect on the liraglutide-induced increase in LC3-II expression and decrease in p62 level(P<0.05).Conclusions:These results indicate that liraglutide exerts neuroprotective action against diabetes-induced hippocampal neuronal injuries and cognitive impairments.The mechanism of these protective effects may relate to elevated autophagy level,which is activated through AMPK/mTOR pathway.